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Epitope delivery system with Escherichia coli heat labile enterotoxin B subunits serving as carriers

A heat-resistant enterotoxin and delivery system technology, applied in the direction of introducing foreign genetic material, antiviral agents, and microbial-based methods using carriers, can solve problems such as the inability to maintain the quality uniformity of the complex and the high price of chemical cross-linking agents

Inactive Publication Date: 2012-09-19
冯强
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, chemical crosslinking agents are expensive and cannot maintain the uniformity of composite quality

Method used

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  • Epitope delivery system with Escherichia coli heat labile enterotoxin B subunits serving as carriers
  • Epitope delivery system with Escherichia coli heat labile enterotoxin B subunits serving as carriers
  • Epitope delivery system with Escherichia coli heat labile enterotoxin B subunits serving as carriers

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] Example 1, construction of pMD18T-LTB-Linker engineering bacteria.

[0085] Using pMD18T-LT as a template, pMD18T-LTB-Seq1, pMD18T-LTB-Seq2, pMD18T-LTB-Seq3, pMD18T-LTB-Seq4, pMD18T-LTB-Seq4-G, pMD18T-LTB-Seq4 were constructed by PCR method -Seq2, pMD18T-LTB-Seq4-Seq3 recombinant plasmids, each plasmid was transformed into E.coli TOP10, and pMD18T-LTB-Linker engineering bacteria were constructed after enzyme digestion and sequencing identification.

[0086] Linker is therefore specifically: Seq1, Seq2, Seq3, Seq4, Seq4-G, Seq4-Seq2 and Seq4-Seq3.

Embodiment 2

[0087] Example 2, construction of engineering bacteria expressing LTB-Linker-Pep1 and LTB-Linker-Pep2.

[0088] Using the pMD18T-LTB-Linker in Example 1 as a template, connect the tested Pep1 and Pep2 fragments to pMD18T-LTB-Linker by PCR to construct pMD18T-LTB-Linker-Pep1 and pMD18T-LTB-Linker-Pep2 recombinant plasmids , using endonucleases NcoI and BamHI to perform double digestion to obtain the target fragment, which was ligated with the same double digestion plasmid pET22b to construct and express recombinant plasmids pET22b-LTB-Linker-Pep1 and pET22b-LTB-Linker-Pep2.

[0089] LTB-Linker-Pep1 includes: LTB-Seq1-Pep1(L1P1), LTB-Seq2-Pep1(L2P1), LTB-Seq3-Pep1(L3P1), LTB-Seq4-Pep1(L4P1), LTB-Seq4-G-Pep1 (L4GP1) and LTB-Seq4-Seq2-Pep1 (L42P1).

[0090] LTB-Linker-Pep2 includes: LTB-Seq1-Pep2(L1P2), LTB-Seq2-Pep2(L2P2), LTB-Seq3-Pep2(L3P2), LTB-Seq4-Pep2(L4P2), LTB-Seq4-Seq2-Pep2 (L42P2) and LTB-Seq4-Seq3-Pep2 (L43P2).

[0091] Transform Escherichia coli E.coli BL21(DE3) wi...

Embodiment 3

[0092] Example 3, the expression of LTB-Linker-Pep1 and LTB-Linker-Pep2.

[0093]Recombinant engineered bacteria pET22b-LTB-Linker-Pep1 / E.coli BL21(DE3) and pET22b-LTB-Linker-Pep2 / E.coliBL21(DE3) were expressed by 1L fermentation respectively, and IPTG was added to the final concentration of 0.1mmol / L, Expression was induced overnight at 18°C. The expression results are shown in the appendix figure 1 and attached figure 2 .

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Abstract

The invention relates to design techniques of an antigenic epitope delivery system and preparation purification techniques, and relates to application of the antigenic epitope delivery system to epitope vaccine development and preparation. An epitope delivery system with Escherichia coli heat labile enterotoxin B subunits serving as carriers is characterized in that Escherichia coli heat labile enterotoxin B (LTB) subunits are connected with specific peptides by means of recombinant DNA (deoxyribose nucleic acid) technology, and then the corresponding B lymphocyte epitope or T lymphocyte epitope is connected to the LTB subunits. According to experiments, the B lymphocyte epitope carried by the antigenic epitope delivery system can effectively impel animals to generate antibodies aiming at the epitope; and the T lymphocyte epitope carried by the antigenic epitope delivery system can effectively impel animals to generate CD8 lymphocyte cells aiming at the epitope. Therefore, the antigenic epitope delivery system can play a significant role in development of epitope vaccines.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to the design and preparation method of an antigen epitope delivery system using Escherichia coli heat-labile enterotoxin B subunit as a carrier, and relates to the application of the system in B-cell epitope vaccines and T-cell epitope vaccines Applications. Background technique [0002] Epitope is a special chemical group in an antigen molecule that determines the specificity of an antigen, also known as an antigenic determinant. cell receptor (BCR) and the basic unit of antibody specific binding. In the immune response, the antigen epitopes recognized by TCR and BCR are different, which are called T cell epitopes and B cell epitopes respectively. The major histocompatibility complex (MHC) is a highly polymorphic cell surface molecule that presents polypeptide fragments to T cells. The antigenic peptide combined with MHC class I molecules is 8-12 amino acids, which is t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/70C12N15/81C12N15/85A61P39/00A61K47/48C12R1/19
Inventor 冯强
Owner 冯强
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